The major components of vegetable oils used in the food industry are the saturated fatty acids, palmitic acid (C16:0) and stearic acid (C18:0), the monounsaturated fatty acid, oleic acid (C18:1) and the polyunsaturated fatty acids, linoleic acid (C18:2) and α-linolenic acid (C18:3). These fatty acids are present in the oils mostly in form of triacylglycerols, in which three fatty acids are esterified to a glycerol molecule. Small amounts of diacyglycerols, monoacylglycerols, phospholipids and free fatty acids may also be present in vegetable oils, along with components such as sterols etc.
The number, position, and conformation of carbon-carbon double bonds in the fatty acid present in the triacylglycerols of the oil influences its physical properties such as melting temperature and other chemical properties as well as its nutritional value, and the applications to which it may be put, particularly in the food industry. For example, the presence of a carbon double bond in a monounsaturated fatty acid or polyunsaturated fatty acid lowers its melting temperature, compared to the melting temperature of a saturated fatty acid of the same carbon chain length, such that the C-18 unsaturated fatty acids, oleic acid, linoleic acid, and linolenic acid, are all liquid at ambient temperature.
Additionally, the susceptibility of a fatty acid to oxidation increases proportionately with the number of carbon double bonds present in the fatty acid molecule, greatly reducing the suitability of oils containing polyunsaturated fatty acids to applications involving the use of prolonged heat in the presence of oxygen, such as cooking and other food service applications, or in non-food applications such as use in the production of cosmetics, pharmaceuticals and candles. For applications that require solid fat components such as in solid cooking fats, shortenings, or margarines, it is necessary to have moderately high levels of saturated fatty acids, or the functionally equivalent trans-fatty acids. Trans-fatty acids have carbon double bonds in the trans-orientation rather than the naturally-occurring cis-orientation.
Currently, many oils high in unsaturated fatty acids are subjected to chemical hydrogenation, to improve their suitability in cooking and food service applications. However, undesirable trans-fatty acids may be produced in this process.
The nutritional quality of vegetable oils depends on the content of both saturated fatty acids and trans fatty acids (Wollett and Dietshy, Amer. J. Clin. Nutr. 60: 991-96, 1994). The contribution of high levels of some saturated fatty acids in the diet, particularly palmitic acid, to increased blood cholesterol, and more particularly to increased low density lipoprotein (LDL), is well-established. Elevated LDL in the blood has been associated with an enhanced risk of cardiovascular disease in humans. Moreover, trans-fatty acids also elevate LDL cholesterol in a manner similar to palmitic acid. However, not all saturated fatty acids are associated with elevated cholesterol. For example, stearic acid is reported to have neutral effects on blood cholesterol (Wollett and Dietshy, 1994 (supra)). In this respect, the high melting temperature of stearic acid (approximately 70° C.) also makes it particularly suitable in solid fat applications. Accordingly, because of its neutral effects on blood cholesterol levels, a high stearic acid-containing oil is a desirable substitute for partially-hydrogenated plant oils currently used in margarine production.
The oil obtained from seeds of the Theobroma cacao plant has an unusual composition that provides several highly desirable properties in the food, particularly confectionary, industries such as chocolate manufacture. The oil typically has about 40% or more stearic acid as well as about 36% oleic acid. More than 50% of the TAG in the oil is oleo-distearin, having one oleic acid molecule and two stearic acid molecules esterified in the TAG. The oil has high levels of palmitic acid in addition to stearic and oleic acids, often esterified on symmetrical triacylglycerols such as sn-1,3 distearoyl sn-2 oleoyl acyl glycerol (SOS), sn-1 palmitoyl sn-2 oleoyl sn-3 stearoyl acyl glycerol (POS), and sn-1,3 dipalmitoyl sn-2 oleoyl acyl glycerol (POP). This composition provides a sharp melting point at about 35° C., with softening at 30-32° C. At only slightly lower temperatures, below about 20° C., the oil is a solid with brittle fracture properties. The oil, also known as “cocoa butter” is in demand and has a high value.
The unreliability and price fluctuation of cocoa butter supply has made the confectionary industry search for a more reliable source of alternative plant fat which can be used as a substitute for cocoa butter. The most common cocoa butter equivalents to date have been made by interesterification of palm oil, palm mid-fraction and oils derived from some tropical oil-bearing plants, such as illipe (Shorea stenoptera), Shea (Butyrospermum parrkii), sal (Shorea robusta) and kokum (Garcinia indica) which contain high level of stearic acid, but insufficient level of palmitic acid. These tropical seed lipids are currently used as the stearic acid donor. The major TAG in the mid-fraction from palm oil is POP and its interesterification with these high-stearic tropical oils using a 1,3-specific lipase can produce TAG's that resemble the fatty acid composition and TAG structures of cocoa butter (Mojovic et al., Enzyme Microb Technol. 15: 438-443, 1993). As these tropical fats are generally expensive, the industry has also been producing cocoa butter substitute through hydrogenation and fractionation of common vegetable oils, including cottonseed oil. In this approach, the undesirable health effect of trans fatty acids resulted from the hydrogenation process are a serious impediment for the industry.
Isolated and purified vegetable oils such as cottonseed oil are composed mostly (>95%) of triacylglycerols (TAGs) that are synthesized and deposited during seed development. TAG molecules consist of three fatty acids esterified to a glycerol backbone at the sn-1, sn-2 and sn-3 positions. Briefly, the de novo biosynthesis of fatty acids in cotton seed, as in other oilseeds, occurs in the stroma of plastids during development and growth of the seeds, ie. before maturation. Fatty acids are then exported from the plastids in the form of acyl-CoA thioesters to the cytoplasmic endomembrane systems (endoplasmic reticulum, ER) where modification of fatty acids occurs after transfer of the acyl groups from the CoA thioesters to phospholipids by acyltransferases. This is followed by TAG assembly and storage in the oleosomes.
The biotin-containing enzyme acetyl-CoA carboxylase (ACCase) catalyses the first committed step in the pathway by activating acetyl-CoA to the three carbon intermediate, malonyl-CoA, by addition of a carboxyl group. The malonyl group is then transferred from CoA to an acyl-carrier protein (ACP), which serves as the carrier for the growing fatty acid chain. Malonyl-ACP is reacted with a second acetyl-CoA condensing enzyme, ketoacyl-ACP synthase III (KASIII), resulting in a four carbon chain. The repeated process of adding two-carbon units on the elongated fatty acid chain is catalyzed by KASI leading to the formation of palmitoyl-ACP. KASII catalyzes the elongation of palmitoyl-ACP to stearoyl-ACP. A soluble stearoyl-ACP Δ9-desaturase introduces the first double bond into stearoyl-ACP to convert it to oleoyl-ACP in the plastid. The extended, saturated fatty acyl chain and the monounsaturated oleate are cleaved off the ACP by a specific thioesterase enzyme, FatB or FatA, respectively, enabling them to exit the plastid and enter the cytoplasm. Saturated fatty acids released into the cytoplasm are not further modified. However, oleic acid can be further modified on the endoplasmic reticulum (ER) membranes by the action of membrane-bound desaturases. Phosphatidylcholine (PC)-bound acyl chains serve as a substrate for ER localized, lipid modifying enzymes, such as fatty acid desaturase 2 (FAD2) which introduces a double bond into oleic acid on the sn-2 position of PC to produce linoleic acid. All the modified and unmodified fatty acyl groups then form a pool while attached to CoA. In cotton, but not in other temperate zone oilseeds, oleic acid may be used as substrate for cyclopropanation catalysed by cyclopropane fatty acid synthase to produce dihydrosterculic acid. This fatty acid is subsequently desaturated to produce sterculic acid and then α-oxidased to produce malvalic acids. Finally fatty acyl groups are incorporated into storage lipids via the Kennedy pathway by the sequential esterification of glycerol-3-phosphate by the action of a series of TAG assembly enzymes.
The enzyme ketoacyl-ACP synthase II (KASII) (EC 2.3.1.41) catalyzes the elongation of palmitoyl-ACP to stearoyl-ACP. The ketoacyl-ACP synthases are often referred to as condensing enzymes of the KAS family. KASI, II and III differ in their chain length specificities, KASI elongates C4:0 to C16:0, while KASII elongates C16:0 to C18:0 (FIG. 1).
Cottonseed oil, which is widely used as a vegetable oil for food applications, produced by conventional (wild-type) upland cotton (G. hirsutum) typically contains approximately 26% palmitic acid (range 22-28%), 1-2% stearic acid, 15% oleic acid (range 13-18%) and 58% linoleic acid (range 52-60%) (Cherry, J. Am. Oil Chem. Soc. 60: 360-367, 1983; O'Brien, Cottonseed Oil. In: F. D. Gunstone (Ed.) Vegetable Oils in Food Technology Composition, Properties and Uses. Blackwell Publishing, Oxford, pp. 203-230, 2002). Unhydrogenated cottonseed oil also contains low levels (0.5-1%) of cyclopropane (CPA) or cyclopropene (CPE) fatty acids, mainly malvalic (MVL), sterculic (STC) and dihydrosterculic acids (DHS) (Shenstone and Vickery, Nature 190: 68-169, 1961; Cherry, 1983 (supra)) These fatty acids accumulate almost exclusively in the embryonic axes of cottonseed. CPA and CPE are not found at detectable levels in major oilseed crops other than cotton, including in palm oil, soybean, corn, canola, mustard, sunflower, safflower, peanut, linseed, other Brassicas etc.
The first committed step to produce these uncommon fatty acids is catalysed by a cyclopropane fatty acid synthase (CPA-FAS) which adds a methylene group across the double bond of oleic acid to produce DHS (FIG. 2). In cotton, most of the DHS is desaturated by the enzyme CPA desaturase to produce STC, most of which is further modified by α-oxidation to form MVL (FIG. 2). MVL is the predominant cyclopropenoid fatty acid in wild-type cottonseed oil.
The relatively high level of saturated fatty acids, mainly palmitic acid, in cottonseed oil compared to oils from most other temperate zone oilseed crops contributes to the oxidative stability of cottonseed oil by offsetting the greater instability of the other, unsaturated fatty acid components. It also imparts the high melting point required for making such products as margarine and shortening. Except for palm oil, cottonseed contains the highest palmitic acid level (26%) among the major commodity vegetable oils. Cottonseed oil also contains a high level of linoleic acid which is oxidatively unstable and therefore limits the shelf life of the oil and makes it unsuitable for some food applications.
Conventional cottonseed oil is therefore often processed by partial hydrogenation during which the polyunsaturated linoleic acid is transformed into more stable monounsaturated (oleic) and saturated (stearic) fatty acids. Partial hydrogenation results in a number of structural changes to a fraction of the fatty acids, including the shifting of a double bond. This may lead to the production of trans fatty acids (TFA) which are isomers of the naturally occurring unsaturated fatty acids, such as elaidic acid which is the trans-isomer of oleic acid. Oleic and elaidic acids contain the same number of atoms (C18:1), with a double bond in the same location, but it is the conformation of the double bond that sets them apart. TAG containing elaidic acid, with the trans double bond configuration, has a much higher melting point than oleic acid. Partial hydrogenation also converts cyclopropanoic or cyclopropenoic fatty acids to branched chain fatty acids by opening up the cyclopropane ring, producing a branched fatty acid with a additional methyl group attached to C9 or C10 of the fatty acid carbon chain.
Compared with polyunsaturated fatty acids, oleic acid is more stable towards oxidation both at ambient storage temperatures and at the high temperatures used in cooking and frying of food. Studies with a number of vegetable oils such as safflower and soybean oils indicate that high-oleic vegetable oils are slower to develop rancidity during storage, or to oxidatively decompose during frying or other use, compared to oils that contain high amounts of polyunsaturated fatty acids (Fuller et al., J. Food Sci. 31: 477-480, 1966; Mounts et al., J. Am. Oil Chem. Soc. 65: 624-628, 1998).
It is known that malvalic and sterculic acids are potent inhibitors of animal Δ9-stearoyl-CoA desaturase. Although the CPA and CPE fatty acids are not stable and are mostly eliminated during oil processing, particularly by hydrogenation, the residual oil in the meal and the whole cottonseed used in the feed industry could exert negative effects on animal health. Feeding farmed animals with excess amounts of cottonseed is thought to possibly cause a number of health problems for animals and may affect the quality of animal products, such as the hardening of fats in egg yolk and milk (Johnson et al., Nature 214: 1244-1245, 1967; Roehm et al., Lipids 5: 80-84, 1970). Methods have been developed to inactivate cyclopropenoid fatty acids through specialised partial hydrogenation processes. Merker and Mattil, 1965 reported a hydrogenation process in which malvalic and sterculic acids were selectively reduced to their dihydro or tetrahydro derivatives, by means of a nickel catalyst, without significant reduction of the linoleic acid or trans acid formation. Hutchins et al., Journal of American Oil Chemists Society 45: 397-399, 1968 showed selective hydrogenation of the cyclopropenoid groups in cottonseed oil by means of a packed-bed reactor and nickel catalysts under milder conditions. However, these hydrogenation processes add additional costs for processing of the oil and are not desirable.
In the 1970's, the cotton breeding program of the Acala SJ series in California (Cherry, 1983 (supra)) reduced palmitic acid from 23.3 to 22.7%, increased oleic acid from 16.6% to 17.3% and reduced total cyclic fatty acids from 0.9% to 0.8% in cottonseed oil. However, compared to achievements in other oilseed crops, these changes were only minor, reflecting the narrow genetic base of elite cotton varieties as a result of persistent selection on traits other than oil quality.
Four different cDNAs encoding FAD2 were isolated from cotton (Liu et al., Australian Journal of Plant Physiology 26: 101-106, 1999a; Liu et al., Plant Physiol. 120: 339, 1999b; Pirtle et al., Biochim. Biophys. Acta 1522: 122-129, 2001, all herein incorporated by reference), among which ghFAD2-1 was determined to play a major role in the production of linoleic acid in cottonseed oil. Analysis of gene expression suggested that the ghFAD2-1 gene was specifically expressed in developing seeds, with maximal expression during the middle maturity stage of seed development (Liu et al., 1999a (supra)).
U.S. Pat. No. 6,974,898 (herein incorporated by reference) describes the generation of cottonseed oil containing up to 77% oleic acid by downregulation of microsomal Δ12 desaturase (FAD2) by RNAi methods. Palmitic acid levels in the oils were reduced.
Furthermore, novel oils having approximately equal proportions of palmitic, stearic and oleic would have considerable potential for use as a cocoa butter substitute.
There is therefore a need to increase the levels of palmitic and stearic acid levels in vegetable oils for particular uses.